We have continued to characterize the cis-regulatory elements of the murine alpha A-crystallin promoter responsible for the lens specific expression of this gene. Hybrid genes containing murine alpha A 5' flanking sequences and the gene coding for the bacterial enzyme chloramphenicol acetyltransferase (CAT) were constructed and their expression studied in explanted chicken lens epithelia and in transgenic mice. Our results indicated the presence of a proximal (-88/+46) and a distal (-111/-88) domain which must interact for promoter function in the explanted chicken lens epithelia. The sequence -88/-60 is essential for promoter function. The distal domain activates the proximal domain when placed at the 5' end but not when inserted at the 3' end of the CAT gene. The distal domain does not activate the enhancerless SV4O promoter. Point mutations indicated that bases at positions -108 and -109 are essential for the activating properties of the distal domain in explanted chicken lens epithelia. Experiments with transgenic mice showed that the sequence -88/+46 directs CAT gene expression specifically to the lens. Gel retardation and methylation interference experiments provided evidence for selective binding of different embryonic chicken lens nuclear proteins to sequences -111/-84 and -83/-55. The protein factor binding -111/-84 may have some similarities to the transcription factor Spl.